The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However the method of constructing an Ad vector containing a foreign liaand in the HI loop of the fiber knob has proved difficult. In this study, eye developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp451 and/or Clal site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the A;a-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)containing peptide motif (as a model) and containing a Csp451 and/or;or Clal recognition site, were ligated into the Csp451 and/or Clal-digested plasmid. The foreign transgene expression cassette was inserted into the Ef deletion site of the vector plasmid and the fiber-mutant ad vector was produced by transfection of the Pacl-digested plasmid into 293 cells, The virus containing the RGB or NC;R peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated C,CAR-independent cell entry pathway: The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.